In-Gel Digestion
Why In-Gel Digest?
Difficult / impossible to extract intact proteins from the gel
Generate peptides that facilitate Protein ID
Enzymes?
Trypsin, Trypsin, Trypsin…--Cleaves after Arg (R) & Lys (K)
Lys C--Cleaves after Lys (K)
V8-- (Cleaves after Asp (D) & Glu (E)
Reduction/Alkylation of Cys (C)?
Necessary for 1D bands and Silver Stained 2D gel plugs
Optional for CBB or Sypro stained 2D gel plugs
The Steps of an In-Gel Digestion
Wash, wash, wash…
alternating 25mM Ammonium Bicarbonate (ABC)
and Acetonitrile (ACN)
Why? …to remove residual SDS & acetic acid
Reduction/Alkylation
ProGest
Reduce with DTT
Alkylate with Iodoactamide
Why? ...to recover Cys containing peptides
Wash Again
Dehydrate with ACN / speedvac
Rehydrate/Incubate with Trypsin
Extract peptides (ACN/Formic Acid)
ProMS
De-salt/concentrate (ZipTips)
& spot to a MALDI target
http://www.genomicsolutions.co.uk/software/
Protein Identification
Peptide Mass Fingerprint (PMF)
Measure the mass of each tryptic peptide
Query a database of known proteins
This can be either a protein database or
a DNA database that undergoes a 6 frame translation
See how many peptides from the experimental data
match to theoretical tryptic peptides of proteins
in the database
Most commonly used database search engines are:
Protein Prospector
http://prospector.ucsf.edu
Mascot
http://www.matrixscience.com
MALDI
Matrix-Assisted Laser Desorption Ionization
Advantages
Rapid analysis/high throughput
Acquisition is easily automated
Disadvantages
Sensitivity not as good as LC/MS/MS
Many (~5-7) peptides required for a confident ID
Protein must be in the database to be identified
Unable to query EST databases
No sequence data is obtained
When to Use MALDI for Protein ID
CBB stained 2D gel plug (Success rate ~99%)
Silver Stained gel plug (Success rate ~ 50-60 %)
the success rate increases as the protein amount increases
Bands from SDS Page gels (and many 2D features) contain more
than one protein, MALDI will ID only the 1-3 most
abundant proteins.
Protein ID by MALDI (peptide mass fingerprint) is only successful
if the protein is in a protein database or DNA database
(Most useful when the genome of the organism is known.)
What is LC/MS/MS?
The digested pool of peptides is injected onto a
reverse phase C18 column. (300 mm to 50mm diameter)
As the peptides elute from the column instead of
being detected by a UV detector they are directly
infused into an Electrospray Ionization (ESI) mass
spectrometer.
The tandem mass spectrometer (Triple Quadrupole, Ion
Trap, or Quadrupole TOF) measures the m/z of the peptides
as they elute, selects a single m/z, then fragments that
peptide and measures the m/z of all the fragment ions.
Protein Identification Using Fragment Ion Data
The mass of each peptide along with the mass of the fragment
ions is used to query the database
A single peptide can provide a confident ID
Because of this many proteins can be identified in a
mixture.
The data can be searched against EST databases
If no protein is identified then the data can be manually
interpreted to get the full length sequence of the peptide
(de novo sequencing)
The sequence data can be used to map post-translational
modifications
LC/MS/MS
Advantages
Can be the most sensitive method for protein ID
(the smaller the column the more sensitive)
Unambiguous search results (very few false positives)
Only a single peptide is required for ID
The ability to perform limited homology searches
single substitution within a peptide
Able to ID proteins from simple mixtures (~20)
such as immunoprecipitates
The data can be searched against EST databases
Disadvantages
Time (Both acquisition and analysis)
Chromatography problems of NanoLC